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UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Recoding and reassignment in protists Author(s) Heaphy, Stephen M. Publication date 2018 Original citation Heaphy, S. 2018. Recoding and reassignment in protists. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2018, Stephen Heaphy. http://creativecommons.org/licenses/by-nc-nd/3.0/ Item downloaded http://hdl.handle.net/10468/6122 from Downloaded on 2021-09-23T15:29:44Z Recoding and reassignment in protists By Stephen Heaphy Thesis in fulfilment for the degree of PhD (Science) National University of Ireland, Cork January 2018 Head of School: Rosemary O’Connor Supervisor: Pavel Baranov Declaration This thesis is my own work and has not been submitted for another degree, either at University College Cork or elsewhere. _______________________________ Stephen Heaphy 1 Acknowledgements The work that went into this thesis and others like it cannot be done by one individual alone. A debt of gratitude is owed to my colleagues, collaborators, friends and family. I would like to thank Pasha Baranov for giving me the opportunity to pursue this peculiar endeavour through his Science Foundation Ireland awarded grant. The atmosphere he created in the lab supported creativity and discovery that I’m sure would it be hard to find elsewhere. His door was always open for light chat and serious discussion. I would like to thank all the members of the lapti lab, in particular Patrick O’Connor and Audrey Michel who were immense help throughout. In addition I would like to thank our colleagues in the recode lab in particular John Atkins who provided much encouragement and advice, along with Gary Loughran and Sinead O’Loughlin. 2 Table of Contents Preface ......................................................................................................................................... 4 Abstract ............................................................................................................................ 10 Introduction ..................................................................................................................... 11 Recoding .......................................................................................................................... 21 Position dependent termination and widespread obligatory frameshifting in Euplotes translation ..................................................................................................................... 21 Diversity of antizyme decoding mechanisms among protists: classical +1 frameshifting, stop codon readthrough and single ORFs ....................................................................... 42 Reassignment ................................................................................................................... 67 Novel ciliate genetic code variants including the reassignment of all three stop codons to sense codons in Condylostoma magnum ........................................................................ 67 Conclusions ...................................................................................................................... 79 References........................................................................................................................ 80 Appendix ........................................................................................................................ 100 3 Preface Proteins are synthesised in the cell by a process known as translation, which is comprised of three different stages; initiation, elongation and termination. Translation initiation in eukaryotes involves the recruitment of the ribosome and initiation complex to an mRNA molecule which is facilitated by the 5’ cap and poly-A tail, ensuring an initiating methionine tRNA is brought to the P-site of the ribosome corresponding to an initiating ATG codon on the mRNA. The ribosome moves by translocating from codon to codon on the mRNA, an aminoacyl- tRNA synthetase charges each tRNA with one of 20 standard amino acid bringing it to the A-site of the ribosome forming a chain. Finally the process of termination is brought about when a class I release factor recognises one of three signals for termination (i.e. stop codons), triggering hydrolysis and releasing the chain of amino acids, i.e. the polypeptide. The polypeptide then folds into a functional protein. These biomolecules are at the core of life as we know it. Their existence in cellular biology is paramount for many purposes including; catalysing metabolic reactions, acting as transport molecules, structural purposes and for DNA replication and repair to name but a few. The synthesis of proteins is an enormously energy expensive process. Due to their extreme importance and high production costs they are also highly regulated. The standard genetic code which comprises 61 amino acid specifying sense codons and three stop codons, was long considered to be unchangeable. Since there are only 20 standard amino acids to choose from, the genetic code expresses a level of redundancy, for example both CAA and CAG codons specify glutamine. A change to the 3rd codon position, i.e. the ‘wobble position’ from A to G or vice versa, in the case of glutamine codons will not change the amino acid being incorporated. The earliest variations to standard decoding were discovered in the 1960’s and 1970’s in bacteria and viruses, where ribosomal frameshifting and stop codon readthrough were identified. It was hypothesised that organisms with small genomes could utilize such ‘recoding’ events to maximise their coding potential. In more recent decades various other decoding anomalies were uncovered such as two additional non-standard amino acids, (selenocysteine and pyrrolysine), translational bypassing and stop codon reassignment. These events are rare in occurrence and are often regulatory in function. For example, the gene orinithine decarboxylase antizyme (OAZ) in 4 eukarotes and release factor 2 in bacteria, both require a frameshift during mRNA translation to synthesise a full length protein. Recent large scale sequencing projects have provided a wealth of new examples of frameshifting in bacteria and other organisms and provide the basis for new repositories such as the Recode Database (Bekaert et al. 2010). Additionally, new techniques make identifying recode events easier. One such recently developed technique is ribosome profiling (Ribo-seq), which captures the ~30nt protected mRNA ‘footprint’ of translating ribosomes, where it is then isolated and sequenced. This concept was hypothesized decades before its subsequent development in the lab of J. Weissman at the University of California, Santa Cruz (UCSF). The first publically available Ribo-seq dataset was published in April 2009, “Genome-Wide Analysis in Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling” (Ingolia et al. 2009). By applying a specific 15nt offset to the 30nt footprint it is possible to identify which codon is in the ribosome A-site of elongating ribosomes. In combination with transcript data (RNA-seq), the level of translation efficiency could be determined (Ribo- seq/RNA-seq). Ribosome profiling provided a novel way of quantifying cellular translation. The practical uses and applications of this new technique were not lost on the scientific community and our lab was one of the first to realise its potential. I commenced my PhD in the summer of 2013 and contributed to the development of a genome browser, Genome Wide Information on Protein Synthesis (GWIPS-viz), which provides analysis and visualization of Ribo-seq data (https://gwips.ucc.ie/). I was involved in developing a pipeline for processing Ribo-seq and RNA-seq data and generating the relevant tracks for each genome. Initially we made Ribo-seq data available from published studies for ten different genomes plus the corresponding RNA-seq. It became apparent that this tool was very useful for identifying recoding events, as large peaks in the profiling data corresponded to pausing ribosomes were observed at frameshift sites of; dnaX from E.coli, the gp gene in Bacteriophage lambda and antizyme 1 (OAZ1) in human and mouse genomes. It also provided evidence for stop codon readthrough in different organisms and contributed to identifying a novel regulatory mechanism in the AMD1 gene in humans. Currently there are 24 genomes including the protists Plasmodium falciparum and Trypanosoma brucei available on GWIPS-viz. 5 Protists, which form an informal grouping of eukaryotes are neither animals, plants nor fungi. They include certain microscopic algae and red algae, which are often included in the Archaeplastida group of eukaryotes. Protists include other eukaryotic supergroups including; Excavata, Amoebozoa, Hacrobia, Apusozoa and certain Opisthokonta. The term protist also includes the very well-studied SAR supergroup, which consists of the superphyla: Stramenopiles, Alveolata and Rhizaria. Stramenopiles are also known as heterokonts, they include a major group of algae commonly known as diatoms, which are routinely used in environmental monitoring. The alveolates include numerous phyla such as: ciliates, dinoflagellates and apicomplexa. The latter phylum includes Plasmodium, the causative agent of malaria. The ciliates are of particular interest. Named for their cilia, they have provided the scientific community with an array of exceptional insights into cell biology. Ciliates contain